Performance of a highly sensitive rapid diagnostic test (HS ......RESEARCH ARTICLE Performance of a highly sensitive rapid diagnostic test (HS-RDT) for detecting malaria in peripheral

RESEARCH ARTICLE

Performance of a highly sensitive rapid

diagnostic test (HS-RDT) for detecting malaria

in peripheral and placental blood samples

from pregnant women in Colombia

Ana Marıa Vasquez1*, Ana Catalina Medina1, Alberto Tobon-Castaño1, Maritza Posada1,

Gabriel Jaime Velez1, Ana Campillo2, Iveth J. Gonzalez2, Xavier Ding2

1 Grupo Malaria, Facultad de Medicina, Universidad de Antioquia, Medellın, Colombia, 2 Foundation for

Innovative New Diagnostics (FIND), Geneva, Switzerland

* [email protected]

Abstract

Background

Pregnancy poses specific challenges for the diagnosis of Plasmodium falciparum infection

due to parasite sequestration in the placenta, which translates in low circulation levels in

peripheral blood. The aim of this study is to assess the performance of a new highly sensi-

tive rapid diagnostic test (HS-RDT) for the detection of malaria in peripheral and placental

blood samples from pregnant women in Colombia.

Methods

This is a retrospective study using 737 peripheral and placental specimens collected from

pregnant women in Colombian malaria-endemic regions. Light microscopy (LM), conven-

tional rapid diagnostic tests (Pf/Pv RDT and Pf RDT), and HS-RDT testing were performed.

Diagnostic accuracy endpoints of LM, HS-RDT and RDTs were compared with nested poly-

merase chain reaction (nPCR) as the reference test.

Results

In comparison with nPCR, the sensitivity of HS-RDT, Pf RDT, Pf/Pv RDT and LM to detect

infection in peripheral samples was 85.7% (95% CI = 70.6–93.7), 82.8% (95% CI = 67.3–

91.9), 77.1% (95% CI = 61.0–87.9) and 77.1% (95% CI = 61.0–87.9) respectively. The sen-

sitivity to detect malaria in asymptomatic women, was higher with HS-RDT, where LM and

Pf/Pv RDT missed half of infections detected by nPCR, but differences were not significant.

Overall, specificity was similar for all tests (>99.0%). In placental blood, the prevalence of

infection by P. falciparum by nPCR was 2.8% (8/286), by HS-RDT was 1% and by conven-

tional RDTs (Pf RDT and Pf/Pv RDT) and LM was 0.7%. The HS-RDT detected placental

infections in peripheral blood that were negative by LM and Pf/Pv RDT, however the number

of positive placentas was low.

PLOS ONE | https://doi.org/10.1371/journal.pone.0201769 August 2, 2018 1 / 14

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OPENACCESS

Citation: Vasquez AM, Medina AC, Tobon-CastañoA, Posada M, Velez GJ, Campillo A, et al. (2018)

Performance of a highly sensitive rapid diagnostic

test (HS-RDT) for detecting malaria in peripheral

and placental blood samples from pregnant

women in Colombia. PLoS ONE 13(8): e0201769.

https://doi.org/10.1371/journal.pone.0201769

Editor: Georges Snounou, Universite Pierre et

Marie Curie, FRANCE

Received: April 23, 2018

Accepted: July 20, 2018

Published: August 2, 2018

Copyright: © 2018 Vasquez et al. This is an open

access article distributed under the terms of the

Creative Commons Attribution License, which

permits unrestricted use, distribution, and

reproduction in any medium, provided the original

author and source are credited.

Data Availability Statement: All relevant data are

within the paper.

Funding: This study has been conducted with

funding from the Departamento Administrativo de

Ciencia, Tecnologıa e Innovacion de Colombia

(Colciencias), grant 111572553345, CT 825-2015

(AMV) (http://www.colciencias.gov.co/). The

funders had no role in study design, data collection

and analysis, decision to publish, or preparation of


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http://creativecommons.org/licenses/by/4.0/

http://www.colciencias.gov.co/

Conclusions

The sensitivity of HS-RDT to detect P. falciparum infections in peripheral and placental sam-

ples from pregnant women was slightly better compared to routinely used tests during ANC

visits and at delivery. Although further studies are needed to guide recommendations on the

use of the HS-RDT for malaria case management in pregnancy, this study shows the poten-

tial value of this test to diagnose malaria in pregnancy in low-transmission settings.

Introduction

Malaria in pregnancy (MiP) is associated with several adverse outcomes in mother, fetus, and

newborn, including maternal anemia, miscarriage, premature delivery and low birth weight

[1]. In the Americas region, a total of 562,800 malaria cases were reported in 2016, 15.3% of

them in Colombia [2]. It is estimated that at least half million women at reproductive age live

in two of the most endemic areas of this South American country (Nariño and Choco) [3],

being at risk of malaria infection.

In Colombia, MiP can be caused by both Plasmodium vivax and Plasmodium falciparum[4–7], the latter being responsible for the most severe consequences [6,8,9]. Early detection

and effective management of infections is crucial for reducing the burden of MiP and for pre-

venting malaria-related pregnancy complications. This is particularly relevant in low-malaria

transmission areas such as Colombia, where intermittent preventive treatment is not recom-

mended [10].

Light microscopy (LM) remains the standard of practice for the diagnosis of suspected

malaria prior to treatment [2]. Nevertheless, LM presents several limitations, including a

reduced sensitivity for detecting low-density infections [11]. It is increasingly appreciated that,

in low-transmission settings, a great proportion of asymptomatic cases are caused by low-den-

sity blood-stage malaria infections [12,13]. In addition, pregnant women frequently present

low-density parasitaemia due to sequestration of P. falciparum in the placenta and their subse-

quent circulation at low-density levels in peripheral blood. Furthermore, the absence of clinical

signs decreases the probability of diagnosing malaria cases which, together with these low- and

fluctuating parasite densities, makes diagnosis by LM challenging [14,15].

Alternatives to LM include the detection of parasite antigens in peripheral blood by rapid

diagnostic tests (RDTs) [16]. RDTs are lateral flow immunochromatographic dipstick assays

that detect either one or more Plasmodium proteins. Antigens detected by commercially avail-

able RDTs include histidine-rich protein 2 (HRP2), aldolase and parasite lactate dehydroge-

nase (pLDH) [16–18]. The use of RDT have facilitated access to malaria diagnosis outside

health facilities in peripheral communities, as less training and investment in infrastructure is

required when compared with LM and molecular techniques [19].

While RDTs have been extensively evaluated for the diagnosis of malaria among non-preg-

nant population, few data exist on the accuracy of this test for screening MiP, especially in

low-transmission settings outside sub-Saharan Africa [19–22]. In these endemic areas, several

studies have shown that RDTs display a sensitivity similar to LM, having both an improved

performance when detecting malaria in symptomatic pregnant women as compared to asymp-

tomatic ones [21,22]. Nonetheless both, LM and RDT, fail to detect a large proportion of low-

density infections identified by polymerase chain reaction (PCR), mainly in asymptomatic

pregnant women screened during antenatal care (ANC) visits [20,21].

HS-RDT for malaria in pregnancy


the manuscript. FIND donated all HS-RDTs used in

the current study.

Competing interests: The authors have declared

that no competing interests exist.


Recently, a new HRP2 specific highly sensitive RDT (HS-RDT) has been made available

(Alere™ Malaria Ag Pf ultra-sensitive RDT). Using the same immunocrhomatographic cassette

platform and volume requirements, the HS-RDT displays about ten-fold lower limit of detec-

tion (LoD, ~80 pg/mL) than current best-in-class RDTs [18,23]. When using blood specimens

from asymptomatic individuals, recent studies showed an improved sensitivity of the HS-RDT

compared with conventional RDT in Myanmar (44% vs. 0%, respectively) and Uganda (84%

vs. 62%), suggesting that the new test could be a useful tool for malaria elimination strategies

[24].

Considering the improvement in LoD, the easiness to use, and the fact that the HS-RDT ful-

fills many of the ASSURED criteria [25] and end-user requirements [26,27], the new test could

be an optimal tool for detecting malaria in pregnant women naturally presenting low-density

infections. The purpose of this study is to evaluate the performance of the new HS-RDT for

the detection of P. falciparum in peripheral and placental blood specimens collected from preg-

nant women in Colombia.

Materials and methods

Study design and population

A retrospective descriptive study was conducted to evaluate the performance of the HS-RDT

for screening malaria in peripheral and placental blood samples from pregnant women, com-

pared with LM and conventional RDTs, and using nPCR as the reference standard. The cur-

rent study used data and samples collected in the frame of a larger cross-sectional project

conducted from May 2016 to September 2017 in Colombia, aimed to characterize asymptom-

atic infections in Colombian pregnant women and to assess the impact of these infections in

malaria-related adverse pregnancy outcomes. A total of 766 pregnant women self-presenting

at local hospitals for ANC visit, at delivery or seeking medical care for suspected malaria were

consecutively recruited. Women, aged� 15 years old, at any gestational age, and living in

peri-urban municipality areas or rural areas with malaria transmission were considered eligi-

ble for the study. Each participant was enrolled only once in one of the two study groups

(ANC or delivery). Overall 737 women, who provided enough samples to perform all labora-

tory testing, were included in the current study.

Ethics statements

All samples were obtained in the frame of a cross-sectional study reviewed and approved by

the Facultad de Medicina Ethics Committee at the Universidad de Antioquia, Medellın,

Colombia (Record 005; 31st March 2016). The approved protocol included the activities per-

formed in the current retrospective study. Before starting any procedure, all participants pro-

vided their written informed consent or informed assent (women <18 years) for the use of

their samples for retrospective testing of malaria parasites. For participants <18 years old addi-

tional informed consent from parents or legal guardians was also obtained. The study was con-

ducted in accordance with the Declaration of Helsinki and local rules and regulations of

Colombia.

Study area

The study was carried out in four Colombian malaria-endemic municipalities: El Bagre, Apar-

tado, Quibdo and Tumaco (Fig 1). El Bagre and Apartado are located in Antioquia’s Depart-

ment, in the northwest of Colombia, with P. vivax as the predominant Plasmodium specie

(spp.) (70%). The municipality of Quibdo is located in the pacific region, in the Choco’s




Department, in the west of Colombia, with P. falciparum as the predominant parasite (80%).

Tumaco is located in the pacific coast in Nariño´s Department, in the southwest of the country

near the border with Ecuador, where the predominant specie is P. falciparum (90%).

Study sites were selected based on the incidence and number of symptomatic cases in gen-

eral population reported during previous years in Colombia [28].

Data and sample collection

Socio-demographic profiles, obstetric history and other clinical data were gathered at local

hospitals using an interview-based questionnaire. Axillary temperature was also recorded

Fig 1. Study area. Map of Colombia showing the four study sites in red dots: Apartado, Bagre, Quibdo and Tumaco.

https://doi.org/10.1371/journal.pone.0201769.g001





using a digital thermometer. Peripheral blood (8 mL) was collected from each participant by

venipuncture, using heparin tubes. When pregnant women were enrolled at delivery, placental

blood (8 mL) was also collected, briefly, a small incision was made on the maternal side of the

placenta, near the cord insertion area after a wash with saline (0.9%), placental blood accumu-

lated at the place of insertion was collected by aspiration with a pasteur pipette.

Sample processing

Blood samples were transferred to the local hospital’s laboratory. Thick and thin blood smears

were prepared (80 μL of blood) and LM readings and Pf/Pv RDT testing (5 μL) were carried

out on-site. Women with positive LM or Pf/Pv RDT results received free antimalarial treat-

ment according to national treatment guidelines. A total of 100 μL of blood were stored at

-80˚C until transport to the reference laboratory at the Universidad de Antioquia in Medellın

for HS-RDT and Pf RDT testing. In addition, 200 μL of blood were spotted on Whatman filter

paper #3 (Fisher, Ref 1003–917), air-dried, and stored at room temperature in sealed bags with

desiccant for nPCR testing in the reference laboratory.

Diagnostic test procedures

Conventional rapid diagnostic test (Pf/Pv RDT). The SD Bioline Malaria antigen Pf/Pv

(Standard Diagnostics, Korea, 05FK80) was performed in local hospitals by trained staff, using

fresh blood samples and according to manufacturer’s instructions. This test–referred through

the document as Pf/Pv RDT–was able to distinguish between P. falciparum, P. vivax and

mixed infections based on the detection of the following antigens: HRP2 and P. vivax-specific

pLDH. The Pf/Pv RDT was selected because it is one of the most commonly used RDT in

Colombia and is one of the top-10 performing HRP2 and pLDH tests for detecting P. falcipa-rum and P. vivax, as reported by the World Health Organization (WHO)-FIND RDT evalua-

tion program [17].

Highly sensitive rapid diagnostic test (HS-RDT). Frozen blood samples were tested ret-

rospectively in duplicate (two test performed by sample) by two independent trained research-

ers using the Alere Malaria Ag P.f RDT Ultra-Sensitive (Standard Diagnostics, Korea,

05FK140); referred here as HS-RDT. This test was performed according to manufacturer’s

instructions. Test results were read after 20 minutes and the outcome was interpreted as

invalid (no control line), positive (presence of the control and P. falciparum line), or negative

(presence of the control line present, but no P. falciparum line). The final result (invalid, posi-

tive or negative) was based on the consensus of the two independent test. In case of discrepant

results the test was repeated a third time to confirm results.

Conventional rapid diagnostic test for quality control (Pf RDT). A second conven-

tional RDT, in addition to the Pf/pv RDT, was used retrospectively in frozen blood samples, in

order to discard antigen degradation due to sample freezing. The SD Bioline Malaria Ag P.f

RDT (Standard Diagnostics, Korea, 05FK50), referred in this manuscript as Pf RDT, was used

in duplicate by two independent trained researchers, according to manufacturer’s instructions.

The Pf RDT is specific for HRP2 antigen and was selected on the basis of its performance in

the WHO-FIND malaria RDT product testing programme [17]. The final result (invalid, posi-

tive or negative) was based on the consensus of the two independent readers. In case of dis-

crepant results the test was repeated to confirm results.

Light microscopy (LM). Field-stained thick and thin blood slides were read by an expert

malaria microscopist according to national guidelines [29]. Parasitaemia was estimated against

200 leukocytes (assuming a standard value of 8,000 leukocytes/μL of blood) and was expressed

as parasites/μL (p/μL). P. falciparum parasitaemia was calculated counting ring forms, while P.




vivax parasitaemia was calculated counting all asexual forms. A sample was considered nega-

tive if after the examination of 200 microscopic fields at 100x magnification, no parasites were

observed. As a quality control, a second reading was performed in all PCR positives samples

and in 10% of negative ones. Discrepant results (positive vs. negative; parasitaemia

difference > 50%; different species) were resolved by a third reading. The final parasitaemia

was the average of all readings, calculated as previously described: parasite densities was calcu-

lated by averaging the two counts and in the blood smears with non-concordant results, para-

site density was calculated by averaging the two most concordant counts [30].

Nested PCR (nPCR). Deoxyribonucleic acid (DNA) was extracted from half blood-spot

filter using QIAamp DNA Mini Kit (Qiagen, Germany, Ref 51306), according to manufac-

turer’s instructions. Nested PCR was performed as a two-step procedure, following the proto-

col described by Singh et. al 1999 with minor modifications [31]. This protocol consists of a

universal PCR followed by nested species-specific PCR to detect the 18S ribosomal gene of P.

falciparum. Each 20 μL reaction mix contained 2 μL of the eluted DNA. Positive and negative

reaction controls were included. Amplification products were resolved in a 1.5% agarose gel

stained with GelRed ™ (Biotium, ref. 41003, United States) and visualized under ultraviolet

light. The current nPCR method was selected as the reference test based on its availability to

detect Plasmodium low-density infections (LoD = 1 p/μL).

Data management and statistical analysis

All data were collected using standardised questionnaires and forms and entered into a Micro-

soft Access database and Excel sheet. Data analysis was carried out using SPSS version 23.0.

Infection prevalence was derived and 95% confidence intervals (CI) were calculated when

applicable. Sensitivity, specificity, positive predictive value (PPV), and negative predictive

value (NPV) were determined for LM (comparator test), Pf/Pv RDT (comparator test), Pf

RDT (comparator test), and HS-RDT (index test), using nPCR as the reference standard.

Kappa coefficient was calculated to assess the agreement among different diagnostic methods,

accounting for random effect. Complementary analysis for diagnostic test accuracy was carried

out in two subgroups of participants (asymptomatic and symptomatic).

Definitions

Low-density infection [32] was defined as Plasmodium spp. infection detected by nPCR but

not by LM. Symptomatic infections were defined as infections detected by LM, HS-RDT, con-

ventional RDTs, or nPCR in pregnant women with fever (axillary temperature� 37.5˚C) or

history of fever in the last 3 days [12]. Asymptomatic infections were defined as infections

detected by LM, HS-RDT, conventional RDTs, or nPCR in pregnant women without fever or

history of fever in the last 3 days [12]. Symptomatic pregnant women were defined as study

participants with fever (axillary temperature� 37.5˚C) or history of fever in the last 3 days,

regardless if they were positive for malaria. Asymptomatic pregnant women were defined as

study participants without fever or history of fever in the last 3 days, regardless they were posi-

tive for malaria.

Results

Study participants baseline characteristics

Overall, 737 pregnant women were recruited across all study sites (Fig 2), 60.8% (448/737) dur-

ing ANC and 39.2% (289/737) at delivery. Socio-demographic characteristics, obstetric and

clinical information, as well as history of malaria are shown in Table 1. The median age of




participants was 23 years (range 15–45), being 41.0% (302/737) primigravida. At enrollment,

3.3% (24/737) of pregnant women presented fever (axillary temperature >37.5˚C) and 7.1%

(52/737) reported history of fever within the last three days. The percentage of participants

reporting history of malaria within current pregnancy was 4.2% (31/737).

Infection rate by diagnostic test

In peripheral blood samples (Table 2), the overall positivity rate detected by the HS-RDT

(4.6%; 34/737) was similar to nPCR (4.7%; 35/737). Thirty infections were observed by Pf RDT

(4.1%; 30/737) and twenty-seven by Pf/Pv RDT and LM (3.7%; 27/737). Parasite density values

obtained by LM were 2,417 rings/μL [interquartile range 729–4,052 rings/μL of blood (range

140–41,211)]. Most infections (30/35 for nPCR) were observed in the ANC group.

Fig 2. Study participant flow and testing results. The chart shows the total number of pregnant women recruited during antenatal care visits and at delivery, the

number of peripheral and placental blood samples collected, as well as the overall number of malaria infections detected by each test. �Discrepant results when

compared with the reference test ANC (antenatal care); Pos. (positive); Neg. (negative); LM (light microscopy); Pf (P. falciparum); Pv (P. vivax); RDT (rapid diagnostic

test); HS-RDT (highly sensitive rapid diagnostic test); nPCR (nested polymerase chain reaction).


Table 1. Baseline characteristics of study participants at enrolment.

Total (N = 737) ANC (N = 448) Delivery (N = 289)

Age (years): median (IQR) 23 (20–28) 23 (19–28) 24 (21–28)

Gestational age (weeks): median (IQR) 30 (17–38) 20 (13–27) 38 (37–39)

Gravidity: median (IQR) 1 (0–2) 1 (0–2) 1 (0–1)

Primigravida: N (%) 302 (41.0) 153 (34.2) 149 (51.6)

Fever at enrollment: N (%) 24 (3.3) 15 (3.3) 9 (3.1)

History of fever (72 hours): N (%) 52 (7.1) 30 (6.7) 22 (7.6)

Malaria within current pregnancy: N (%) 31 (4.2) 20 (4.5) 16 (5.5)

ANC (antenatal care); IQR (Interquartile range); N (sample size).

https://doi.org/10.1371/journal.pone.0201769.t001






Regarding placental specimens (Fig 2), the overall infection rate, as determined by nPCR,

was 2.8% (8/286). The percentage of P. falciparum positive cases detected by the HS-RDT was

1% (3/286). LM, Pf/Pv RDT and Pf RDT showed a positivity rate of 0.7% (2/286). The parasi-

taemia of the two placental positive samples detected by LM was 160 and 200 rings/μL of

blood. Low-density infections were observed in both, peripheral (1.1% 8/737) and placental

(2.1%; 6/286) blood.

Performance of diagnostic tests in peripheral blood

Using nPCR as the reference standard (Table 3), the HS-RDT showed slightly better sensitivity

[85.7% (95% CI = 70.6–93.7)], when compared with LM [77.1% (95% CI = 61.0–87.9)], conven-

tional Pf/Pv RDT [77.1% (95% CI = 61.0–87.9)], and Pf RDT [82.8% (95% CI = 67.3–91.9)], but

the differences were no significant. Both LM and Pf/Pv were highly specific (100.0%). Pf RDT and

HS-RDT displayed few false positive results when compared with nPCR. The false positive rate

for Pf RDT and HS-RDT, defined as the percentage of all negatives with a positive result, was

0.14% (95% CI = 0.0–0.8) and 0.57% (95% CI = 0.2–1.5), respectively.

Regarding test performance in symptomatic (61) and asymptomatic (649) pregnant

women, all methods showed sensitivity greater than 95% and specificity of 100% when detect-

ing symptomatic infections (21/61) (Table 4). There was one symptomatic case not detected

by any of the three HRP2-based RDTs, but confirmed by LM (4,018 rings/μL) and nPCR. In

asymptomatic participants, LM and Pf/Pv RDT missed half of the infections detected by nPCR

(14/30). Although Pf RDT and HS-RDT also missed some cases, the HS-RDT showed higher

Table 2. P. falciparum positivity rate by diagnostic test in peripheral blood samples collected during ANC visits and at delivery.

Peripheral blood; N (%) Total (N = 737) ANC (N = 448) Delivery (N = 289)

LM 27 (3.7) 27 (6.0) 0 (0.0)

Pf/Pv RDT 27 (3.7) 27 (6.0) 0 (0.0)

Pf RDT 30 (4.1) 27 (6.0) 3 (1.0)

HS-RDT 34 (4.6) 29 (6.5) 5 (1.7)

nPCR 35 (4.7) 30 (6.7) 5 (1.7)

Low-density infections 8 (1.1) 3 (0.7) 5 (1.7)

ANC (antenatal care); LM (light microscopy); Pf (P. falciparum); Pv (P. vivax); RDT (rapid diagnostic test); HS-RDT (highly sensitive rapid diagnostic test); nPCR

(nested polymerase chain reaction); N (sample size).


Table 3. Accuracy of LM, Pf/Pv RDT, Pf RDT, and HS-RDT for the diagnosis of P. falciparum infections in peripheral blood of pregnant women.

PCR Value (95% CI)

(+) (-) Total Sensitivity Specificity PPV NPV Kappa

LM (+) 27 0 27 77.1% 100.0% 100.0% 98.9% 0.9

(-) 8 702 710 (61.0–87.9) (99.5–100.0) (87.5–100.0) (97.8–99.4) (0.8–1.0)

Pf/Pv RDT (+) 27 0 27 77.1% 100.0% 100.0% 98.9% 0.9

(-) 8 702 710 (61.0–87.9) (99.5–100.0) (87.5–100.0) (97.8–99.4) (0.8–1.0)

Pf RDT (+) 29 1 30 82.8% 99.9% 96.7% 99.1% 0.9

(-) 6 701 707 (67.3–91.9) (99.2–100.0) (83.3–99.4) (98.2–99.6) (0.8–1.0)

HS-RDT (+) 30 4 34 85.7% 99.4% 88.2% 99.3% 0.9

(-) 5 698 703 (70.6–93.7) (98.5–99.8) (73.4–95.3) (98.3–99.7) (0.8–1.0)

LM (light microscopy); Pf (P. falciparum); Pv (P. vivax); RDT (rapid diagnostic test); HS-RDT (highly sensitive rapid diagnostic test); nPCR (nested polymerase chain

reaction); (+) (positive); (-) (negative); PPV (positive predictive value); NPV (negative predictive value); CI (confidence interval)







sensitivity [71.4% (CI 95% = 45.4–88.3)] compared with LM and Pf/Pv RDT [50% (CI 95% =

27.8–73.2)], but differences were no significant. Overall, 27 participants with no data on tem-

perature or fever history were excluded for this analysis.

Performance of diagnostic tests in placental blood

Using nPCR in placental blood as the reference test, the sensitivity of HS-RDT was slightly

higher [37.5% (95% CI = 13.7–69.4)], compared with LM and conventional RDTs [25.0%

(7.1–59.1)]. The HS-RDT detected one additional infection (3/8) compared with LM and con-

ventional RDTs (2/8). Using nPCR in placental blood as the reference standard, neither LM

nor Pf/Pv RDT were able to detect any placental infection in peripheral blood, while nPCR,

HS-RDT and Pf RDT detected 62.5% (95% CI = 30.6–86), 37.5% (95% CI = 13.7–69.4), and

25% (95% CI = 7.1–59.1) of placental infections respectively. However the number of infected

placentas detected by the reference test was so low (n = 8), that it did not allow to draw conclu-

sions from such a small number of positives.

Discussion

To the extent of our knowledge, this is the first study evaluating the accuracy of the new

HS-RDT for the detection of P. falciparum infections in samples from pregnant women in

Table 4. Performance of LM, Pf/Pv RDT, Pf RDT, and HS-RDT for detecting P. falciparum in peripheral blood of symptomatic and asymptomatic pregnant

women.

Value (95% CI)

Symptomatic (N = 61) Test PCR Sensitivity Specificity PPV NPV

(+) (-)

LM (+) 19 0 95.2% (77.3–99.2) 100.0% (91.2–100.0) 100.0% (83.9–100.0) 97.6% (87.4–99.6)

(-) 0 42

Pf/Pv RDT (+) 18 0 95.2% (77.3–99.2) 100.0% (91.2–100.0) 100.0% (83.9–100.0) 97.6% (87.4–99.6)

(-) 1 42

Pf RDT (+) 18 0 95.2% (77.3–99.2) 100.0% (91.2–100.0) 100.0% (83.9–100.0) 97.6% (87.4–99.6)

(-) 1 42

HS-RDT (+) 18 0 95.2% (77.3–99.2) 100% (91.2–100.0) 100.0% (83.9–100.0) 97.6% (87.4–99.6)

(-) 1 42

Asymptomatic (N = 649) LM (+) 7 0 50.0% (26.8–73.2) 100.0% (99.4–100.0) 100.0% (64.6–100.0) 98.8% (97.7–99.4)

(-) 7 635

Pf/Pv RDT (+) 7 0 50.0% (26.8–73.2) 100% (99.4–100.0) 100% (64.6–100.0) 98.8% (97.7–99.4)

(-) 7 635

Pf RDT (+) 9 0 64.3% (38.8–83.7) 100.0% (99.4–100.0) 100.0% (70.1–100.0) 99.2% (98.1–99.7)

(-) 5 635

HS-RDT (+) 10 3 71.4% (45.4–88.3) 99.5% (98.6–99.8) 76.9% (49.7–91.8) 99.4% (98.3–99.7)

(-) 4 632

LM (light microscopy); Pf (P. falciparum); Pv (P. vivax); RDT (rapid diagnostic test); HS-RDT (highly sensitive rapid diagnostic test); nPCR (nested polymerase chain

reaction); (+) (positive); (-) (negative); PPV (positive predictive value); NPV (negative predictive value); CI (confidence interval); N (sample size).






low-transmission settings. Using nPCR as the reference standard, the HS-RDT showed a

slightly improved sensitivity for detecting malaria in peripheral and placental specimens, com-

pared with LM and conventional RDTs. This improvement was particularly evident when

screening asymptomatic participants and for identifying low-density parasite infections. In

addition, the study demonstrates the need of further investigations of usefulness of the new

test for detecting placental infections in peripheral blood using a large sample of placental

infections.

Overall, 4.7% of the study participants were found to harbor malaria parasites in peripheral

blood. This observation was in line with recent studies conducted in Colombia, showing a

prevalence of P. falciparum mono-infections of 3.0–4.1% [6,7]. When using nPCR as the refer-

ence test, HS-RDT displayed a slightly improvement in sensitivity (85.7%), compared with LM

(77.1%) and conventional RDTs (77.1–82.8%). Similar results have been reported in non-preg-

nant population, where HS-RDT sensitivity was higher than RDT in both, high- and low-

transmission settings [24].

As previously reported, conventional RDTs performed similar to LM when detecting

peripheral P. falciparum infections in pregnant women [15,19–22,33,34]. Accuracy discrepan-

cies between the two conventional RDTs used in this study could be explained by the different

panel detection scores observed in the WHO-FIND malaria RDT testing programme (95% Pf

RDT vs. 92% Pf/Pv RDT for P. falciparum) [35]. Furthermore both, LM and conventional

RDTs, missed a higher number of low-density infections than nPCR. This observation empha-

sized the underestimation of malaria infections by tests routinely used in pregnant women

attending ANC visits and at delivery. In this sense, further studies are needed to guide recom-

mendations on the use of the HS-RDT in pregnant women with inherently low-density P. fal-ciparum levels.

The accuracy of the HS-RDT for screening P. falciparum infections in symptomatic and

asymptomatic women was also evaluated in the current study. In agreement with previous

findings, the performance of conventional RDTs and LM was superior in symptomatic than

asymptomatic cases, showing high sensitivity (>95%) and specificity (100%), probably due to

higher parasite densities in febrile infections [21,33]. Nevertheless, the HS-RDT showed an

improved sensitivity in afebrile participants (71.4%), where LM and conventional RDTs

missed 50% of infections. Although the clinical relevance of pregnant women as asymptomatic

parasite reservoir is not yet understood, asymptomatic carriers could remain as reservoirs for

malaria transmission [9,11]. In this sense, the HS-RDT could be a promising tool for managing

MiP during ANC visits and at delivery, particularly in low-transmission settings where inter-

mittent preventive treatment during pregnancy is not recommended and only positive malaria

cases are treated [19].

It is worth noting that one of the symptomatic cases detected by LM in Apartado was nega-

tive by all three RDTs regardless being a specimen with high parasite density. This result sug-

gests the potential presence of pfhrp2/pfhrp3deletions, as previously reported in the south of

Colombia and in the border of Peru [36,37]. Certainly, the current configuration of the

HS-RDT does not allow identifying parasites with pfhrp2 deletions, which might be one of the

current limitations of the test.

Although sensitivity is a key feature to ensure end-users that the HS-RDT is unlikely to

miss any malaria infection, the PPV is also an important parameter to take into account. Over-

all, the HS-RDT showed lower PPV compared with LM (88% vs. 100%), especially in afebrile

women. This observation could be related to the presence of false positive results, probably

due to HRP2 persistence in blood [19,24,38] or cross-reaction with other proteins such as

rheumatoid factor [16,24,39]. Further research is needed to evaluate the HRP2 HS-RDT-based

positivity time-course in MiP, as well as potential cross-reactivity limitations.




Regarding placental specimens, the overall infection rate confirmed by nPCR of 2.8% was

lower than in peripheral blood samples and in line with latest prevalence reported in the area

[6]. Given that access to placental tissue and blood during pregnancy is practically impossible,

an ideal diagnostic test should be able to detect placental infections in peripheral blood. The

current results showed that all test performed in peripheral blood underestimated placental

infections detected by nPCR, probably due to parasite sequestration in placenta [40]. In the

study areas, the routine tests used for screening malaria in peripheral blood (i.e. LM and Pf/Pv

RDT) failed to detect all placental infections. Instead, the HS-RDT and Pf RDT improved the

diagnosis of placental malaria, detecting 37.5% and 25% infections, respectively, however the

low number of infected placentas make it difficult to draw any conclusion. In light with the

above findings, further studies are needed to assess the usefulness of the new HS-RDT for

detecting placental malaria.

Study limitations

The low number of malaria-positive samples was the main limitation of this study. Sample size

was estimated to meet the main objective of a larger cross-sectional project aiming to charac-

terize asymptomatic infections in Colombian pregnant women and to assess their impact in

malaria-related adverse pregnancy outcomes. Despite the sample size for the current retro-

spective sub-study was too small to provide statistically significant accuracy values, HS-RDT

displayed an improved sensitivity compared with LM and RDTs for screening MiP. Future

studies should consider a larger sample size.

While study staff errors cannot be excluded at local hospitals, training was provided before

starting the study, making operator factors an unlikely cause of poor LM and Pf/Pv RDT per-

formance. Moreover, the HS-RDT was performed in duplicate, in parallel to a quality control

test (Pf RDT), and in a reference laboratory by highly trained staff. However, the fact that

HS-RDT was run in laboratory conditions with expert readers does not necessary illustrates

performance under field conditions. In this regard, further studies showing the usefulness of

the HS-RDT detecting MiP in remote settings, outside reference centers are needed. Finally,

the low prevalence of malaria cases in this study did not allow an evaluation of the impact of

missed infections in pregnancy and birth outcomes.

Conclusion

The improved sensitivity of the HS-RDT over LM and conventional RDTs for detecting gesta-

tional and placental malaria, particularly in asymptomatic women, indicates the potential

value of this test for managing malaria in pregnancy. This performance should be confirmed

by larger studies under field conditions, using finger-prick blood specimens. The cost-effec-

tiveness of integrating this new tool in the maternal health programs in low-transmission set-

tings should also be evaluated. Finally, the impact that this highly sensitive tool may have in

malaria-related adverse pregnancy and birth outcomes should be investigated.

Acknowledgments

We would like to thank all women and their families for participating in the study. We also

thank the technicians who contributed to participant’s care and data collection, as well as the

municipalities of Apartado, Bagre, Quibdo, and Tumaco.

Author Contributions

Formal analysis: Ana Marıa Vasquez.




Funding acquisition: Ana Marıa Vasquez, Alberto Tobon-Castaño.

Investigation: Ana Catalina Medina, Maritza Posada, Gabriel Jaime Velez.

Methodology: Ana Catalina Medina, Maritza Posada, Gabriel Jaime Velez.

Supervision: Ana Marıa Vasquez, Alberto Tobon-Castaño.

Writing – original draft: Ana Marıa Vasquez, Ana Campillo.

Writing – review & editing: Ana Campillo, Iveth J. Gonzalez, Xavier Ding.

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